Method

Purpose

DNA Construct

Desired Effect

Variations

 

Expression reporting

Homologous genomic regions flanking in-frame reporter gene and selectable marker

Disruption of endogenous gene and expression of reporter gene under control of the endogenous gene’s promoter

Various reporter genes (e.g., lacZ), green fluorescent protein, luciferase

Targeted mutagenesis via homologous recombination in embryonic stem cells

 

DNA electroporation into embryonic stem cells

Point mutations

Homologous genomic regions with desired point mutation flanking lox-P-flanked selectable marker

Replacement of homologous region and removal of selectable marker

Different recombinase systems possible

Conditional mutations

Homologous genomic regions containing lox-P site in intron, flanking lox-P-flanked selectable marker

Produce mutation by removing regions flanked by lox-P sites at desired time

Mutation produced at different times or in specific tissues

Conditional restoration

Homologous genomic regions with lox-P-flanked selectable marker

Restore mutated gene function by removing selectable marker at desired time

Function restored at different times or in specific tissues

Knockin

Homologous genomic regions of gene 1 flanking coding regions of gene 2

Replacement of coding region of gene 1 with that of gene 2 to misexpress gene 2 in expression domain of gene 1

 



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